The primary goal of this proposal is to elucidate the cellular and molecular mechanisms by which the chemokine receptor CXCR4 is regulated. CXCR4 dysregulation has been associated with several pathologies including breast cancer, HIV, WHIM syndrome and cardiovascular disease sates such as chronic ischemic heart disease, angina and human end-stage heart failure. Understanding the mechanisms regulating CXCR4 may provide new strategies for the prevention and treatment of the pathologies associated with CXCR4 dysregulation. CXCR4 undergoes agonist-dependent ubiquitination at the plasma membrane mediated by the E3 ubiquitin ligase AIP4, which targets the receptor for degradation in lysosomes by serving as an endosomal sorting signal. However, insight into the cellular and molecular mechanisms by which AIP4 recognizes and ubiquitinates activated CXCR4 and its role on CXCR4 signaling is lacking. We provide initial evidence demonstrating that AIP4 interacts with non-visual arrestins and that phosphorylation may play a role in targeting CXCR4 to lysososmes. We hypothesize that AIP4-mediated ubiquitination of CXCR4 is preceded by phosphorylation leading to arrestin recruitment, which serves as an adaptor to in turn recruit AIP4 to CXCR4. We also provide evidence that suggests that the ubiquitin moiety on CXCR4 not only serves as an endosomal sorting signal, but may also serve as a rapid and immediate terminator of signaling at the plasma membrane. We propose a comprehensive series of studies aimed at examining the interaction between arrestins and AIP4 in cells and in vitro using GST pull-down assays, co- immunoprecipitation studies, siRNA analysis and immunofluorescence microscopy experiments. Our objective is to define the cellular and molecular mechanisms that determine how AIP4 recognizes and ubiquitinates activated CXCR4 to regulate its signaling. The following Specific Aims are proposed: 1. To define how the ubiquitin ligase AIP4 recognizes and ubiquitinates activated CXCR4. 2. To determine the role of phosphorylation in mediating AIP4-dependent ubiquitination and degradation of CXCR4. 3. To determine the role of AIP4-dependent CXCR4 ubiquitination on regulation of CXCR4 mediated signaling.